Copper-dependent proteins with ferroxidase activity play an important role in the mobilization of iron stores from hepatocytes for use by other tissues. Ceruloplasmin, a copper containing secretory glycoprotein synthesized mainly, but not exclusively by hepatocytes, possesses ferroxidase activity and accelerates iron efflux from hepatocytes. The process by which ceruloplasmin performs this function has not been clearly elucidated, nor has the potential role of other cellular proteins in this process has been explored. To determine the role of copper in iron metabolism we will focus our initial studies on iron efflux from hepatocytes. We plan to test our hypothesis that hepatocellular iron transport is facilitated by cellular proteins with copper-dependent ferroxidase activity. We present preliminary evidence from a unique animal model, the Long-Evans Cinnamon (LEC) rat that lacks the functional copper-transporter atp7b. LEC rats have an increased hepatic iron content, decreased rates of hepatocellular iron efflux, and decreased hepatocellular membrane associated ferroxidase activity compared the normal parental strain, Long-Evans Agouti. These data suggest the acquisition of copper by a cellular ferroxidase involved in iron efflux from hepatocytes is dependent upon the function of the intracellular copper transporting ATPase, atp7b. Other preliminary data suggest the presence of plasma membrane ferroxidase activity in HuH7, a human hepatoblastoma cell line. In this study, we initially plan to explore the mechanism by which ceruloplasmin enhances iron efflux from hepatocytes by determining the site of action of this protein targeted to specific cellular sites. We will next determine the effect of modulating cellular copper levels of cellular copper-dependent ferroxidase activity and on iron efflux. Finally, we will identify hepatocellular proteins with copper-dependent ferroxidase activity and determine their role in iron efflux. These studies will elucidate the role of copper-proteins in hepatocellular iron export and open new avenues of investigation into the cellular mechanisms governing the metabolism of these two essential metals.